Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1

Abstract

Amyotrophic lateral sclerosis (ALS) involves motor neuron degeneration, skeletal muscle atrophy, paralysis, and death. Mutations in Cu,Zn superoxide dismutase (SOD1) are one cause of the disease. Mice transgenic for mutated SOD1 develop symptoms and pathology similar to those in human ALS. To understand the disease mechanism, we developed a simple behavioral assay for disease progression in mice. Using this assay, we defined four stages of the disease in mice expressing G93A mutant SOD1. By studying mice with defined disease stages, we tied several pathological features into a coherent sequence of events leading to motor neuron death. We show that onset of the disease involves a sharp decline of muscle strength and a transient explosive increase in vacuoles derived from degenerating mitochondria, but little motor neuron death. Most motor neurons do not die until the terminal stage, approximately 9 weeks after disease onset. These results indicate that mutant SOD1 toxicity is mediated by damage to mitochondria in motor neurons, and this damage triggers the functional decline of motor neurons and the clinical onset of ALS. The absence of massive motor neuron death at the early stages of the disease indicates that the majority of motor neurons could be rescued after clinical diagnosis.

Fig. 1.

The definition of L5 lateral ventral horn where neuronal numbers were quantified was derived as follows. Perpendicular to the midline M, line Npassing the central canal was drawn. Parallel to line Nand across point B was line L. PointB was the first point on the contour of the ventral gray matter (tracing from the midline) at which the contour made a >180° turn. Connecting point C (the cross-point of lineN and the contour of gray matter) and pointD (at one-third the distance from point Bto A), line P was drawn. All neurons within the area encompassed by line P and the contour of ventral horn (hatched area) were drawn and measured.

Fig. 2.

Four stages of muscle strength change in G93A mice. The time that mice were capable of hanging onto a wire was measured as an indication of muscle strength. A, Age-dependent decline of muscle strength in G93A mice. Groups of mice at different ages were tested. The number of mice in each age group ranged from 3 to 20. G93A, Mutant SOD1 mice;WT, nontransgenic (wild-type) mice; WS, wild-type SOD1 transgenic mice. Error bars indicate SEM.B, Highly variable onset of muscle weakness among different G93A mice. Each trace represents measurements from one animal. For clarity, only measurements around the RD stage in 20 animals are shown. C, Synchronized plot of muscle strength decline in individual G93A mice. Zero week represents the time point just before the decline begins. For clarity, only 10 traces are shown. D, Average time course of muscle strength decline in G93A mice (n = 12). PMW, Pre-muscle weakness stage; RD, rapid declining stage;SD, slow declining stage; Para., paralysis stage. Error bars represent SEM.

Fig. 3.

Axonal degeneration in L5 ventral roots at different stages of ALS in G93A mice. Plastic sections (1 μm) were stained with toluidine blue. A, WT; B, WS; (C–H), G93A at the following stages: (C) PMW, (D) RD, (E–G) 20, 33, and 50 d after the onset of RD, respectively; H, paralysis.

Fig. 4.

Axon size distribution in L4 and L5 ventral roots. The distribution of axons in the two roots was similar, and therefore the measurements were pooled together in one plot. Three animals were measured for each plot. The three PMW animals (C) were between 120 and 160 d old and therefore were near the RD stage. A–H, Same as in Figure 2.

Fig. 5.

Motor axons undergo atrophy before degeneration.A, Total number of axons in L4 and L5 ventral roots at different disease stages. B, Number of axons larger than 4.5 μm in diameter at different disease stages. C, Number of axons smaller than 4.5 μm in diameter at different disease stages. D, Number of degenerating axons in L4 and L5 ventral roots at different disease stages. Asterisksindicate significance level in comparison with the WT using two-tailed Student’s t test. One asteriskrepresents p < 0.05; two asterisksrepresent p < 0.01. n = 3 for all stages.

Fig. 6.

Changes in ventral horn at different disease stages. Plastic sections (1 μm) were stained with toluidine blue.A–H, Same as in Figure 2.

Fig. 7.

Changes in spinal cord at different disease stages. A, Changes in the number of neurons in the ventral horn. All numbers were averages from two animals and were normalized to the average number of neurons in each wild-type mouse (145 neurons). Filled bars, Total number of neurons;open bars, number of neurons with diameters <25 μm;shaded bars, number of neurons with diameters >25 μm. Note that the division of large and small neuronal groups at 25 μm diameter is arbitrary because there was not a clear division of large and small neuron peaks in the size distribution (data not shown). Changes in vacuole number (B) and vacuole size (C) at different disease stages. Two ventral horns were measured in each animal. The small number of vacuoles in the WT mice are capillary blood vessels that could not be distinguished from the real vacuoles under the light microscope.Asterisks indicate significance level in comparison with the WT, using two-tailed Student’s t test. One asterisk represents p < 0.05; two asterisks represent p < 0.01.n = 3 for all stages.

Fig. 8.

Mitochondrial abnormalities at PMW (A–E) and RD (F) stages.A, A swollen dendritic mitochondrion with dilated cristae (asterisk) and leaking outer membrane (arrow). B, Swollen dendritic mitochondria with dilated and disorganized cristae. A synaptic terminal on the dendrite contains normal mitochondria (arrowhead). C, A proximal dendrite containing mitochondria with broken outer membranes (arrows). Adjacent synaptic terminals contain normal mitochondria (arrowheads). D, Early vacuoles in a proximal axon. Arrows point to mitochondrial remnants. E, Early vacuoles in a dendrite.Arrows point to mitochondrial remnants.F, Massive dendritic vacuolation at the RD stage.

Fig. 9.

Sequence of pathological events leading to motor neuron death in G93A mice. All the data points were derived from the same set of animals. Each data point represents the average of three animals. All the values are normalized against the highest measure (100%) in the sequence. For the hanging test, only the last hanging times before the animals were killed are shown. Error bars have been shown in Figures 3 and 6 and are omitted here for clarity.